Peptide Macrocyclization Assisted by Traceless Turn Inducers Derived from Ugi Peptide Ligation with Cleavable and Resin-Linked Amines

Article abstract of DOI:10.1021/acs.orglett.7b01761

A multicomponent approach enabling the installation of turn-inducing moieties that facilitate the macrocyclization of short and medium-size oligopeptides is described. The strategy comprises the Ugi ligation of peptide carboxylic acids and isocyanopeptide

Full text of DOI:10.1021/acs.orglett.7b01761

Letter
pubs.acs.org/OrgLett
Peptide Macrocyclization Assisted by Traceless Turn Inducers
Derived from Ugi Peptide Ligation with Cleavable and Resin-Linked
Amines
Alfredo R. Puentes,†,‡,§ Micjel C. Morejon,†,‡,§ Daniel G. Rivera,*,†,‡ and Ludger A. Wessjohann*,†
́
†Department of Bioorganic Chemistry, Leibniz Institute of Plant Biochemistry, Weinberg 3, 06120 Halle/Saale, Germany
‡Center for Natural Products Research, Faculty of Chemistry, University of Havana, Zapata y G, 10400 Havana, Cuba
*
S Supporting Information
ABSTRACT: A multicomponent approach enabling the
installation of turn-inducing moieties that facilitate the
macrocyclization of short and medium-size oligopeptides is
described. The strategy comprises the Ugi ligation of peptide
carboxylic acids and isocyanopeptides in the presence of
aldehydes and acid or photolabile amines followed by
cyclization and cleavage of the backbone N-substituents to
render canonical cyclopeptides. Implementing the approach on
solid phase with the use of Rink amide resins led to a new class of backbone amide linker strategy.
he head-to-tail cyclization of short peptides stands as one o1f
the most challenging procedures in synthetic chemistry.
inducers. The approach comprises the ligation of a peptide
T
carboxylic acid and an isocyanopeptide by the Ugi four-
component reaction (Ugi-4CR) using a cleavable amine (or a
resin amine linker), thus leading to a peptide including an N-
alkylated amino acid midway along the sequence. An aldehyde as
fourth Ugi component defines the newly formed α-amino acid
side chain. Our endeavor was to use the known turn-inducing
capability of this N-alkylated peptide fragment to facilitate the
cyclization both in solution and on-resin.
Typical problems encountered in this process are the C-terminal
1
epimerization and cyclooligomerization. Whereas new coupling
2
1,3
reagents and synthetic tools have been developed to partially
solve these issues, they are intrinsically dependent on both the
sequence and the ring size and, therefore, difficult to generalize in
short peptides. Besides the classic solutions of either conducting
the peptide macrocyclization at extreme dilution (even 0.01
4
mM) or employing pseudo-dilution conditions, the most
We have previously documented that N-alkylated pentapep-
successful strategy has been the incorporation of turn-inducing
elements capable of facilitating the macrocyclic ring closure by
12
tide derived from Ugi reactions are relatively easy to cyclize and
1a
that, in the case of a bulk N-substituent, the acyclic peptide occurs
bringing both termini closer.
12a
in a ß-turn conformation. As a result, we envisioned that the
Ugi reaction could be a straightforward way to install acid- and
Owing to their capacity to favor the cis-amide bond, proline
and N-methyl amino acids are well-known turn-inducing
elements that improve peptide cyclization yields when
8
,9
photolabile N-substituents capable of improving the cycliza-
tion efficiency, thus acting as traceless macrocyclization
assistants. Scheme 1 depicts the use of 2,4-dimethoxybenzyl-
1
,5
embedded midway along the acyclic precursor.
The
6
7
incorporation of D-amino acids and pseudo-prolines, i.e.,
13
(
4S)-oxazolidine-4-carboxylic acids derived from serine and
amine (1) in the multicomponent ligation of dipeptide acids
and isocyanides to produce N-alkylated pentapeptides 2 and 4 in
very good yields. Subsequent deprotection of both termini
followed by macrolactamization at 1 mM concentration with
threonine, into linear peptides also makes the macroyclization
much more efficient. Nonetheless, the employment of these
structural elements to assist the peptide cyclization is limited to
the presence of such amino acids in the target cyclic peptide. A
more versatile strategy is the use of a traceless turn inducer, i.e., a
moiety that can be installed in the middle of any peptide to bend
its backbone and be subsequently cleaved or transformed into a
native peptide structure after cyclization. Remarkable examples
14
either propanephosphonic acid anhydride (T3P, known for
minimizing C-terminal epimerization) or PyBOP afforded cyclic
peptides 3a and 5a. The DMB N-substituent was subsequently
removed under acidic conditions to afford canonical peptides 3b
and 5b in good yields of isolated products over four steps. The
use of a prochiral oxo component leads to a ca. 1.5:1 mixture of
diastereomers due to the poor stereoselectivity of the Ugi-4CR,
which is perhaps the only drawback of this strategy.
8
are the use of the photolabile 2-hydroxy-6-nitrobenzyl N-
9
substituent, the acid-labile dimethoxybenzyl (DMB) and tert-
10
butyloxycarbonyl (Boc) N-substituents, as well as dehydro-
1
1
phenylalanine as traceless turn inducers capable of assisting the
cyclization of short peptides.
Herein, we describe a novel strategy for the solution- and solid-
phase macrocyclization of peptides assisted by traceless turn
Received: June 10, 2017
©
XXXX American Chemical Society
A
DOI: 10.1021/acs.orglett.7b01761
Org. Lett. XXXX, XXX, XXX−XXX

Organic Letters
Letter
Scheme 1. Macrocyclization of Pentapeptides Assisted by a
Traceless Turn-Inducing Core Derived from the Ugi Reaction
oNB cleavage protocol produced the cyclic peptide 9b in 48%
yield after HPLC purification. HPLC/ESI-MS analysis after
DMB cleavage showed 75% of purity for the cyclic monomer,
with only 4% of the C-terminal epimer. On the other hand,
HPLC traces of a parallel macrocyclization with the non-N-
alkylated analogue showed 51% of purity of cyclopeptide 9b, with
7
% of the dimer and 9% of the C-terminal epimer. Again, the Ugi-
derived fragment improved the cyclization efficiency as
compared with the canonical peptide and reduces cyclo-
dimerization. It should be mentioned that these examples
encompass the ligation of tripeptide carboxylic acids and amino
acid-derived isocyanides, which move the N-alkylation position
away from the favorable middle and place it one amino acid
toward the C-terminus. However, even if this is not the ideal
positioning, the turn inducer produced better results than
without it.
After proving the efficacy of the method with pentapeptides,
we extended the scope to tetra- and hexapeptides, with the
former ones being very challenging due to their cyclodimeriza-
tion propensity. Scheme 3 depicts the cyclization of tetrapeptide
Scheme 3. Macrocyclization of Tetra- And Hexapeptides
Assisted by a Traceless N-Alkylated Ugi Reaction Fragment
To assess the macrocyclization improvement provided by the
N-substituted Ugi reaction fragment, we also carried out the
T3P-mediated cyclization of the model peptide H-Val-Leu-Gly-
Gly-Leu-OH and compared both results. This study proved that
at 1 mM concentration and 12 h of reaction the cyclization of the
non-N-alkylated peptide rendered 21% yield of cyclic peptide 3b,
while the deprotected peptide 2 led to 52% yield of isolated 3b.
HPLC/ESI-MS analysis of the crude product also showed the
presence of ca. 8% of the unreacted linear precursor, but no
dimer was detected.
We next evaluated the efficacy of a photolabile N-substituent
and the scope of a macrolactonization protocol. As shown in
Scheme 2, N-alkylated peptide 6 was produced by an Ugi
Scheme 2. Macrocyclization of N-Alkylated (Depsi)peptides
Including Traceless Acid- and Photolabile N-Substituents
10 and hexapeptides 13, previously produced in good yields by
Ugi-4CR. As peptide 10 has two Gly in its sequence, we
intentionally introduced the bulky Ile at the N-terminus so that
the cyclization would not be so much facilitated by sequence and
an accurate assistant effect could be assessed. Cyclization of 10
followed by DMB cleavage rendered cyclic tetrapeptide 11b and
the cyclodimer 12b in 39% and 17% yield, respectively, after
HPLC purification (see the Supporting Information). Nonethe-
less, our method did prove much more effective than the
cyclization of the analogous model peptide H-Ile-Phe-Gly-Gly-
OH, which led almost exclusively to the formation of the dimer in
its acyclic and cyclic variants. Analytical HPLC traces of the crude
cyclization showed a monomer/dimer ratio of 2.6:1 for N-
alkylated peptide 10 and a ratio of 1:19 for cyclization of the non-
N-alkylated one.
As expected, cyclization of deprotected hexapeptide 13
proceeded smoothly to furnish cyclic peptide 14b in 61% yield
after DMB/tBu cleavage and purification. Only 4% of the C-
terminal epimer was detected by analytical HPLC/ESI-MS.
In our opinion, the highest potential of the multicomponent/
turn induction strategy to assist peptide cyclization lies on its
possible adaptation to the solid phase. Over the years, much
reaction in good yield and subsequently subjected to
saponification and standard macrolactonization with EDC.
Final DMB cleavage led to isolated cyclic depsipeptide 7b in
6
8% yield over three steps. Alternatively, 2-nitrobenzylamine
15 16
(oNB) and phenylalanine-derived Nenajdenko isocyanide
were employed in the multicomponent synthesis of the N-
alkylated peptide 8, which was subsequently deprotected and
macrocyclized with T3P for 12 h. In this case, the cyclization/
B
DOI: 10.1021/acs.orglett.7b01761
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Organic Letters
Letter
Scheme 4. Solid-Phase Synthesis of Cyclic Peptides by a Multicomponent Backbone Amide Linker (BAL) Strategy
effort has been devoted to develop effective protocols for the on-
resin head-to-tail cyclization of peptides that do not have
trifunctional amino acids such as Glu, Asp, Lys, His, and Ser,
whose side chain are commonly linked to the resin leaving the
two termini available for cyclization. A major advance in this
field was the development of the so-called backbone amide linker
diketopiperazine (DKP) formation, which is common in BAL
approaches if Fmoc deprotection is used at the dipeptide stage.
18
In addition, our multicomponent strategy preserves high
flexibility for protecting groups, as either ethyl or allyl esters
can be used at the C-terminus and Boc/tBu groups at the side
chains, being orthogonal to the Fmoc methodology employed
for peptide elongation.
17
(
BAL) strategy, introduced independently by Albericio and
1
8
19
Barany and Ellman. These approaches enable the on-resin C-
terminal modification and cyclization of peptides anchored to the
resin by an internal amide N-substituent. The protocol
comprises the attachment of an acid-labile substituted
benzaldehyde to the polymeric support, followed by reductive
amination and acylation to incorporate two amino acids.
The scope of this multicomponent BAL strategy was assessed
using oligopeptides ranging from four to seven amino acids. As
shown in Scheme 4, the use of the Rink amide polystyrene resin
MBHA with a loading of 0.71 mmol/g led to the cyclic
pentapeptide 15a in only 14% yield, while the dimer 15b was
isolated in 36% yield. Alternatively, when a Tentagel S RAM resin
with a lower loading (0.26 mmol/g) was used, only the cyclic
pentapeptide (monomer) 16 was isolated in good overall yield.
However, using the same Tentagel S RAM resin, a mixture of
cyclic tetrapeptide (monomer) 17a (18%) and the dimer 17b
(39%) was obtained, proving the difficulty of cyclizing
tetrapeptides without cyclodimerization. To avoid that, perhaps
a resin with a much lower loading is required, as it has been
20
Herein, we extend this multicomponent strategy to the
synthesis of backbone amide-linked peptides and their
subsequent derivatization by peptide growth and final cycliza-
tion. As illustrated in Scheme 4, our approach does not require
the initial incorporation of a cleavable aldehyde linker and
subsequent reductive amination and acylation, but instead it
comprises the direct incorporation of an Fmoc-amino acid and
an isocyanide peptide or amino acid to the polymer support
having the Rink amide linker. To carry out the on-resin Ugi-4CR,
an aminocatalytic transimination step using paraformaldehyde
and piperidine is required prior addition of the acid and the
17
proven in previously reported works.
The on-resin macrocyclization of hexapeptides proceeded
smoothly to furnish cyclic peptides 18 and 19 in good overall
yield and without cyclodimerization. Peptide 18 derives from the
initial incorporation of Fmoc-Ile-OH and isocyanide CN-Phe-
Gly-OMe, while the synthesis of 19 begins with Fmoc-Phe-OH
and ethyl isocyanoacetate. To further prove the scope of this
method, we undertook the total synthesis of the natural product
2
1
isocyanide. Other aldehydes can be employed without such
transimination step but may result in a final mixture of two
diastereomers. As at least a tripeptide is attached to the resin in
the first step, this enables bypassing the difficult acylation step of
the secondary amine and, even more important, avoids
22
crassipin B. This cyclic heptapeptide was produced in overall
C
DOI: 10.1021/acs.orglett.7b01761
Org. Lett. XXXX, XXX, XXX−XXX

Organic Letters
3% yield through the initial multicomponent attachment of
Letter
5
(4) Macrocyclizations under pseudo-dilution conditions comprise the
use of syringe pumps; see, for example: (a) Malesevic, M.; Strijowski, U.;
Fmoc-Phe-OH and ethyl isocyanoacetate to the resin, followed
by peptide growing using the Fmoc strategy and consecutive
macrocyclization and acidic cleavage from resin. In summary, this
new multicomponent BAL strategy can be considered as a
valuable and efficient improvement in peptide synthesis. Several
factors such as the low synthetic cost for incorporating the first
three or four amino acids into the resin, the great tolerance of
protecting groups, and the complete absence of DKP formation,
among others, make it a plausible alternative to classic BAL
approaches. It is especially valuable for cyclic peptides having at
least one Gly or Aib residue or for the construction of D/L
peptide library including natural and non-natural amino acids, as
higher aldehydes can be used leading to the two diastereomers.
In conclusion, we have developed an efficient strategy to assist
peptide macrocyclization both in solution and on the solid phase.
The approach uses the versatile Ugi-4CR for the ligation of
peptides and the simultaneous incorporation of a removable N-
alkyl substituent that serves as a turn-inducing moiety and
facilitates the macrocyclic ring closure. Its assistance to the
macrocyclization was proven with a variety of tetra- to
heptapeptides. An extension of this concept to the solid phase
led to the development of a new BAL strategy, relying for the first
time on the multicomponent incorporation of at least three
amino acids in one step instead of the three on-resin steps
required in traditional BAL protocols for attaching a dipeptide
fragment. Owing to its synthetic prospects, this concept can be
very useful for the peptide, combinatorial, and medicinal
chemistry communities.
Bac
A.; Kreye, O.; Rivera, D. G. Angew. Chem., Int. Ed. 2017, 56, 3501.
5) (a) Chatterjee, J.; Mierke, D. F.; Kessler, H. Chem. - Eur. J. 2008, 14,
508. (b) Favre, M.; Moehle, K.; Jiang, L.; Pfeiffer, B.; Robinson, J. A. J.
Am. Chem. Soc. 1999, 121, 2679. (c) Takeuchi, Y.; Marshall, G. R. J. Am.
Chem. Soc. 1998, 120, 5363. (d) Rothe, M.; Steffen, K.-D.; Rothe, I.
Angew. Chem., Int. Ed. Engl. 1965, 4, 356.
(6) (a) Kessler, H.; Haase, B. Int. J. Pept. Protein Res. 1992, 39, 36.
(b) Yongye, A. B.; Li, Y.; Giulianotti, M. A.; Yu, Y.; Houghten, R. A.;
Martínez-Mayorga, K. J. Comput.-Aided Mol. Des. 2009, 23, 677.
̈
hle, D.; Sewald, N. J. Biotechnol. 2004, 112, 73. (b) Wessjohann, L.
(
1
̈
(7) (a) Dumy, P.; Keller, M.; Ryan, D. E.; Rohwedder, B.; Wohr, T.;
Mutter, M. J. Am. Chem. Soc. 1997, 119, 918. (b) Skropeta, D.; Jolliffe, K.
A.; Turner, P. J. Org. Chem. 2004, 69, 8804. (c) Sayyadi, N.; Skropeta,
D.; Jolliffe, K. A. Org. Lett. 2005, 7, 5497. (d) Fairweather, K. A.; Sayyadi,
N.; Luck, I. J.; Clegg, J. K.; Jolliffe, K. A. Org. Lett. 2010, 12, 3136.
(
8) Meutermans, W. D. F.; Bourne, G. T.; Golding, S. W.; Horton, D.
A.; Campitelli, M. R.; Craik, D.; Scanlon, M.; Smythe, M. L. Org. Lett.
003, 5, 2711.
2
(9) (a) El Haddadi, M.; Cavelier, F.; Vives, E.; Azmani, A.; Verducci, J.;
Martinez, J. J. Pept. Sci. 2000, 6, 560. (b) van Maarseveen, J. H.; Horne,
W. S.; Ghadiri, M. R. Org. Lett. 2005, 7, 4503.
(
10) Cavelier-Frontin, F.; Achmad, S.; Verducci, J.; Jacquier, R.; Pepe,
G. J. Mol. Struct.: THEOCHEM 1993, 286, 125.
11) Le, D. N.; Riedel, J.; Kozlyuk, N.; Martin, R. W.; Dong, V. M. Org.
Lett. 2017, 19, 114.
12) (a) Rivera, D. G.; Vasco, A. V.; Echemendía, R.; Concepcion
Perez, C. S.; Gavín, J. A.; Wessjohann, L. A. Chem. - Eur. J. 2014, 20,
3150. (b) Barreto, A. F. S.; Vercillo, O. E.; Birkett, M. A.; Caulfield, J.
(
(
́
, O.;
́
1
C.; Wessjohann, L. A.; Andrade, C. K. Z. Org. Biomol. Chem. 2011, 9,
5024. (c) Vercillo, O. E.; Andrade, C. K. Z.; Wessjohann, L. A. Org. Lett.
2
008, 10, 205.
ASSOCIATED CONTENT
Supporting Information
(13) For the use of DMB amine in Ugi reactions, see: (a) Shaw, A. Y.;
Xu, Z.; Hulme, C. Tetrahedron Lett. 2012, 53, 1998. (b) Plant, A.;
Thompson, P.; Williams, D. M. J. Org. Chem. 2009, 74, 4870.
(14) T3P is a registered trademark. Original report: (a) Wissmann, H.;
Kleiner, H.-J. Angew. Chem., Int. Ed. Engl. 1980, 19, 133. (b) Review:
Llanes García, A. L. Synlett 2007, 2007, 1328.
■
*
S
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.orglett.7b01761.
Experimental procedures, NMR, and ESI-MS spectra of
selected intermediates and final cyclic peptides (PDF)
(15) Sung, K.; Chen, F.-L.; Huang, P.-C. Synlett 2006, 2006, 2667.
(16) Zhdanko, A. G.; Nenajdenko, V. G. J. Org. Chem. 2009, 74, 884.
(17) (a) de Leon Rodriguez, L.; Weidkamp, A. J.; Brimble, M. A. Org.
AUTHOR INFORMATION
Corresponding Authors
Biomol. Chem. 2015, 13, 6906. (b) Alcaro, M. C.; Sabatino, G.; Uziel, J.;
Chelli, M.; Ginanneschi, M.; Rovero, P.; Papini, A. J. Pept. Sci. 2004, 10,
■
*
*
2
18. (c) Alsina, J.; Rabanal, F.; Giralt, E.; Albericio, F. Tetrahedron Lett.
E-mail: dgr@fq.uh.cu.
E-mail: wessjohann@ipb-halle.de.
1994, 35, 9633. (d) Kates, S. A.; Sole, N. A. Tetrahedron Lett. 1993, 34,
́
1549. (e) Mazur, S.; Jayalekshmy, P. J. Am. Chem. Soc. 1979, 101, 677.
(18) (a) Jensen, K. J.; Songster, M. F.; Vagner, J.; Alsina, J.; Albericio,
ORCID
F.; Barany, G. In Peptides − Chemistry, Structure and Biology: Proceedings
of the Fourteenth American Peptide Symposium (1995); Kaumaya, P. T. P.,
Hodges, R. S., Eds.; Mayflower Scientific: Kingswinford, U.K., 1996; pp
Daniel G. Rivera: 0000-0002-5538-1555
Author Contributions
3
0−32. (b) Jensen, K. J.; Alsina, J.; Songster, M. F.; Vagner, J.; Albericio,
§
A.R.P. and M.C.M. contributed equally.
F.; Barany, G. J. Am. Chem. Soc. 1998, 120, 5441. (c) Alsina, J.; Yokum,
T. S.; Albericio, F.; Barany, G. J. Org. Chem. 1999, 64, 8761.
Notes
(
19) (a) Boojamra, C. G.; Burow, K. M.; Ellman, J. A. J. Org. Chem.
995, 60, 5742. (b) Boojamra, C. G.; Burow, K. M.; Thompson, L. A.;
Ellman, J. A. J. Org. Chem. 1997, 62, 1240.
(20) For reviews, see: (a) Gongora-Benítez, M.; Tulla-Puche, J.;
The authors declare no competing financial interest.
1
ACKNOWLEDGMENTS
D.G.R. is grateful to the Alexander von Humboldt Foundation
for an Experienced Researcher Fellowship.
■
́
Albericio, F. ACS Comb. Sci. 2013, 15, 217. (b) Boas, U.; Brask, J.;
Jensen, K. J. Chem. Rev. 2009, 109, 2092. (c) Alsina, J.; Jensen, K. J.;
Albericio, F.; Barany, G. Chem. - Eur. J. 1999, 5, 2787.
(
21) (a) Morales, F. E.; Garay, H. E.; Mun
Otero-Gonzalez, A. J.; Reyes Acosta, O.; Rivera, D. G. Org. Lett. 2015,
7, 2728. (b) Morejon, M. C.; Laub, A.; Westermann, B.; Rivera, D. G.;
Wessjohann, L. A. Org. Lett. 2016, 18, 4096.
22) Jia, A. Q.; Tan, N. H.; Yongping, Y.; Wu, S. G.; Wang, L. Q.; Zhou,
J. Act. Bot. Sin. 2004, 46, 625−630.
̃
oz, D. F.; Augusto, Y. E.;
REFERENCES
■
(
1) (a) White, C. J.; Yudin, A. K. Nat. Chem. 2011, 3, 509. (b) Lambert,
J. N.; Mitchell, J. P.; Roberts, K. D. J. Chem. Soc. Perkin Trans. 1 2001,
1
́
4
(
(
71.
2) (a) El-Faham, A.; Albericio, F. Chem. Rev. 2011, 111, 6557.
b) Montalbetti, C. A. G. N.; Falque, V. Tetrahedron 2005, 61, 10827.
3) (a) Davies, J. S. J. Pept. Sci. 2003, 9, 471. (b) Humphrey, J. M.;
Chamberlin, A. R. Chem. Rev. 1997, 97, 2243.
(
(
D
DOI: 10.1021/acs.orglett.7b01761
Org. Lett. XXXX, XXX, XXX−XXX